Protein Splicing: From the Foundations to the Development of Biotechnological Applications.

Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.

Structural Insights into Subunits Assembly and the Oxyester Splicing Mechanism of Neq pol Split Intein.

Split inteins are expressed as two separated subunits (N-intein and C-intein) fused to the corresponding exteins. The specific association of both intein subunits precedes protein splicing, which results in excision of the intein subunits and in ligation, by a peptide bond, of the concomitant exteins. Catalytically active intein precursors are typically too reactive for crystallization or even isolation. Neq pol is the trans-intein of the B-type DNA polymerase I split gene from hyperthermophile Nanoarchaeum equitans. We have determined the crystal structures of both the isolated NeqN and the complex of NeqN and NeqC subunits carrying the wild-type sequences, including the essential catalytic residues Ser1 and Thr+1, in addition to seven and three residues of the N- and C-exteins, respectively. These structures provide detailed information on the unique oxyester chemistry of the splicing mechanism of Neq pol and of the extensive rearrangements that occur in NeqN during the association step.

Severe necrosis of oesophageal and gastric mucosa in fatal methanol poisoning.

Methanol is a potent neurotoxic substance that causes severe metabolic acidosis and serious neurological disorders. Most of the cases are accidental exposures to drinking beverages contaminated with methanol. There are few articles reporting pure methanol intoxication; however, it is well known that small quantities of pure methanol causes blindness and death, the minimum lethal dose being 50-100 ml.A case report is presented of a 67-year-old woman, who committed suicide by ingestion of 500 ml of absolute methanol. Despite symptomatic and supportive intensive care, the woman died 23 h after hospital admission due to metabolic acidosis and multiple organ dysfunction syndrome. A complete medico-legal autopsy was performed. Grossly, there was complete detachment of the oesophagus mucosa and brownish discolouration of the gastric mucosa. Histological findings showed diffuse haemorrhagic necrosis of the stomach mucosa and intense acute inflammatory infiltration of the lamina propria. To our knowledge, this is the first autopsy report of such severe digestive injuries. A discussion and review of the recent literature on the subject are given.

Deciphering the role of the thermodynamic and kinetic stabilities of SH3 domains on their aggregation inside bacteria.

The formation of insoluble deposits by globular proteins underlies the onset of many human diseases. Recent studies suggest a relationship between the thermodynamic stability of proteins and their in vivo aggregation. However, it has been argued that, in the cell, the occurrence of irreversible aggregation might shift the system from equilibrium, in such a way that it could be the rate of unfolding and associated kinetic stability instead of the conformational stability that controls protein deposition. This is an important but difficult to decipher question, because kinetic and thermodynamic stabilities appear usually correlated. Here we address this issue by comparing the in vitro folding kinetics and stability features of a set of non-natural SH3 domains with their aggregation properties when expressed in bacteria. In addition, we compare the in vitro stability of the isolated domains with their effective stability in conditions that mimic the cytosolic environment. Overall, the data argue in favor of a thermodynamic rather than a kinetic control of the intracellular aggregation propensities of small globular proteins in which folding and unfolding velocities largely exceed aggregation rates. These results have implications regarding the evolution of proteins.